The major goal of the proposed studies is to apply the current wealth of knowledge about the molecular structure and function of diphtheria toxin (DT) to create genetically inactivated cross- reactive mutant forms of the toxin (CRMs) that will serve as ideal components of future vaccines. The crystallographic structure of DT was solved recently, revealing the topography of the 3 functionally distant domains-the C (catalytic), T (transmembrane), and R (receptor-binding) domains. Using known substitution and deletion mutations that selectively abrogate individual functions, we will seek to define a limited set of CRMs that are appropriate for developing various types of vaccines. First, as an alternative to standard formal intoxoid, the investigators will seek to develop a holotoxin CRM with negligible biologic activity and with minimal alteration in antigenicity and immunogenicity. Second, toxin fragments corresponding to the 3 domains and selected inter- and intra-domain peptide sequences will be investigated as potential immunogens. Third, the investigators will investigate the hypothesis that disruption of the receptor-binding or membrane-translocation functions may alter the immunogenicity of the molecule. Fourth, to create a new vehicle for polysaccharides in conjugate vaccines, the investigators will introduce specific functional residues within CRMs to generate coupling sites for polysaccharides. Fifth, to identify mutant forms of DT appropriate for incorporation into live-vectored vaccines, the investigators will prepare and test multiply mutated CRMs that have suitably low probabilities of reversion. These studies will enhance understanding of the role of each of the critical vaccine antigens which will be: biologically inactive and stable without chemical toxoiding, highly consistent from batch to batch, inexpensively purified by simple affinity chromatography methods, suitable for chemical coupling to bacterial polysaccharide antigens in defined locations, and suitable for insertion into live vectors or for creation of chimeric proteins. The approaches developed in these studies may be applicable to other important vaccine antigens, such as tetanus toxin, pertussis toxin, and other pertussis antigens.